Methods and compositions for multiplex amplification of nucleic acids

ABSTRACT

A method is described for predetermining ratios of primer pairs present in a single reaction vessel so as to achieve approximately equimolar yield of products. The ratios are determined as a function of the length of the amplicon and the length of other amplicons being simultaneously tested. The primers may desirably be for p53 gene sequences.

RELATED APPLICATION

[0001] This application claims priority to U.S. Provisional Application,Serial No. 60/050,405, filed on Jun. 20, 1997, the text of which isexpressly incorporated herein.

BACKGROUND OF THE INVENTION

[0002] The polymerase chain reaction (PCR) is a simple and versatilemethod to amplify in vitro a specific segment of DNA for subsequentstudy (Saiki et al., Science 230:1350 (1985); Saiki et al, Science235:487 (1985)). The PCR method has gained widespread use in biomedicalresearch, and has revolutionized the accurate and early diagnosis ofmany inherited and acquired genetic disorders (Eisenstein, N. Engl J.Med. 322:178 (1990)), particularly those caused by point mutations orsmall insertions or deletions including sickle cell anemia (Saild et al,Science 230:1350 (1985)), hemophilia A (Kogan et al, N. Engl J. Med.317:985 (1987)), Tay-Sach's disease (Myerowitz, Proc. Natl. Acad. Sci.USA 85:3955 (1988); Myerowitz et al, J. Biol. Chem. 263:18587 (1988)),cystic fibrosis (Riordan et al., Science 245:1066 (1989)), and manyothers. With PCR, it is also possible to detect heterozygotic carriersin recessive disorders.

[0003] Polymerase chain reaction (PCR) is used for a variety ofpurposes. PCR can be used to amplify genomic DNA or other sources ofnucleic acids for analysis. It is often desirable to be able to achieveequimolar yields of different length amplicons when performing multiplexPCR or multiple PCR reactions. Having an approximately equimolar yieldof amplicons is particularly useful, for example, when approximatelyequal representation of certain regions of genomic DNA amplified aftermultiplex PCR is desired. Prior to the methods of present invention,finding the appropriate experimental conditions useful to achieve thisresult has been difficult because PCR amplifies nucleic acids havingdifferent lengths with different efficiencies. The yield of longeramplicons is often less than the yield of shorter amplicons because ofthose differences in PCR amplification efficiency. FIG. 1 shows thedifference in yields that one might expect, for example, when startingwith equal primer concentrations used to amplify amplicons of varyinglengths: A, B, C. There is a continuing need in the art for methodswhich permit the amplification of different sequences with the sameefficiency so that approximately equimolar products result.

SUMMARY OF THE INVENTION

[0004] It is an object of the present invention to provide a method ofperforming multiplex PCR which achieve approximately equimolar products.

[0005] It is another object of the invention to provide a set of primersfor amplification of p53.

[0006] It is yet another object of the invention to provide a set ofprimers for amplification of p53 to achieve approximately equimolarproducts.

[0007] It is still another object of the invention to provide a mixtureof primers for performing multiplex PCR.

[0008] These and other objects of the invention are provided by one ormore of the embodiments provided below. In one embodiment of theinvention a method of performing multiple polymerase chain reactions ina single vessel is provided. The method comprises the steps of primingDNA synthesis on a template in a vessel with at least two sets ofprimers. The primers are present in the vessel at a predetermined ratiowhich is described by the formula:

C _(A) =C _(L)(L _(A) ÷L _(L))²

[0009] C_(A) is the concentration of primers for an amplicon A. C_(L) isthe concentration of primer for the longest amplicon. L_(A) is thelength of the amplicon A. L_(L) is the length of the longest amplicon.

[0010] Another embodiment provided by the invention is a method ofperforming multiple polymerase chain reactions in a single vessel. Themethod comprises priming DNA synthesis on a genomic p53 template in avessel with ten sets of primers which amplify exons 2-11 of p53. Theprimers are shown in SEQ ID NO: ID NOS: 1-20. The primers are present inthe vessel at the following ratio: exon 2 (89.4): exon 3 (26.9): exon4(450): exon 5 (245.8): exon 6 (138.3): exon 7 (101.8): exon 8 (193.0):exon 9 (70.8): exon 10 (146.5): exon 11 (177.3).

[0011] According to still another embodiment of the invention a set ofprimers for performing multiple polymerase chain reactions in a singlevessel is provided. The set comprises twenty primers having sequences asshown in SEQ ID NO: 1-20.

[0012] According to yet another embodiment of the invention a mixture ofprimers for performing multiplex polymerase chain reaction is provided.The primers are present in the mixture at a predetermined ratio to eachother. The ratio of the concentrations of the primers is described by:

C _(A) =C _(L)(L _(A) ÷L _(L))²

[0013] wherein C_(A) is the concentration of primers for an amplicon A;wherein C_(L) is the concentration of primer for the longest amplicon;wherein L_(A) is the length of the amplicon A; and wherein L_(L) is thelength of the longest amplicon.

[0014] The present invention thus provides the art with a method usefulfor performing multiplex PCR. This method is particularly useful foramplification of multiple exons of p53. Moreover, a particular primerset useful for performing such multiplex PCR is also provided.

BRIEF DESCRIPTION OF THE DRAWINGS

[0015]FIG. 1 shows the difference in yields that one might expect, forexample, when starting with equal primer concentrations used to amplifyamplicons of varying lengths: A, B, C.

[0016]FIG. 2 illustrates the relationship for given values X andL_(L),using the amplicons from different exons of the human p53 gene asan example.

DETAILED DESCRIPTION OF THE INVENTION

[0017] It is a discovery of the present invention that approximatelyequimolar yields of amplicons of varying lengths can be easily producedby multiplex PCR. It has been determined that varying the primerconcentrations as a function of the lengths of amplicons yieldsapproximately equimolar amounts of amplicons of varying lengths. Therelationship between primer concentration and the length of amplicons isas follows:

C _(A) =C _(L)(L _(A) /L _(L))^(X)

[0018] wherein C_(A)=the concentration of primers for an amplicon A;

[0019] C_(L)=the concentration of primer for the longest amplicon;

[0020] L_(A)=the length of amplicon A;

[0021] L_(L)=the length of the longest amplicon; and

[0022] X is usually not zero and is often between one and three.

[0023] This relationship can be placed in a computer readable medium orbe used with a computer system if desired.

[0024]FIG. 2 illustrates the relationship for given values X and L_(L),using the amplicons from different exons of the human p53 gene as anexample. Using primer concentrations as set forth, for example in FIG.2, one skilled in the art can determine the optimum set of primerconcentrations to yield approximately equimolar yields of varying lengthamplicons in a multiplex or multiple PCR. Preferably, primers havingboth comparable base composition and comparable melting temperatures areused. Also preferably, Mg⁺² concentration, annealing temperatures, andcycling times of the PCR are optimized prior to choosing the desired setof primer concentrations in accordance with the present invention.

[0025] PCR techniques applicable to the present invention include interalia those discussed in PCR PRIMER: A LABORATORY MANUAL, Dieffenbach, C.W. and Dveksler, G. S., eds., Cold Spring Harbor Laboratory Press(1995).

[0026] The present application further provides primer sequences, primerconcentrations, and experimental conditions useful in the amplificationof the coding region of the human p53 gene. Particularly useful primersfor amplification of exons of the p53 gene are set forth in Table 1.TABLE 1 p53 Primer Set 20 primers in 1 mM Tris-HC1, pH 7.4, 0.1 mM EDTA,sequences: Exon 2: 5′-TCATGCTGGATCCCCACTTTTCCTCTTG-3′5′-TGGCCTGCCCTTCCAATGGATCCACTCA-3′ Exon 3:5′-AATTCATGGGACTGACTTTCTGCTCTTGTC-3′ 5′-TCCAGGTCCCAGCCCAACCCTTGTCC-3′Exon 4: 5′-GTCCTCTGACTGCTCTTTTCACCCATCTAC-3′5′-GGGATACGGCCAGGCATTGAAGTCTC-3′ Exon 5:5′-CTTGTGCCCTGACTTTCAACTCTGTCTC-3′ 5′-TGGGCAACCAGCCCTGTCGTCTCTCCA-3′Exon 6: 5′-CCAGGCCTCTGATTCCTCACTGATTGCTC-3′5′-GCCACTGACAACCACCCTTAACCCCTC-3′ Exon 7:5′-GCCTCATCTTGGGCCTGTGTTATCTCC-3′ 5′-GGCCAGTGTGCAGGGTGGCAAGTGGCTC-3′Exon 8: 5′-GTAGGACCTGATTTCCTTACTGCCTCTTGC-3′5′-ATAACTGCACCCTTGGTCTCCTCCACCGC-3′ Exon 9:5′-CACTTTTATCACCTTTCCTTGCCTCTTTCC-3′5′-AACTTTCCACTTGATAAGAGGTCCCAAGAC-3′ Exon 10:5′-ACTTACTTCTCCCCCTCCTCTGTTGCTGC-3′5′-ATGGAATCCTATGGCTTTCCAACCTAGGAAG-3′ Exon 11:5′-CATCTCTCCTCCCTGCTTCTGTCTCCTAC-3′ 5′-CTGACGCACACCTATTGCAAGCAAGGGTTC-3′

[0027] Table 2 shows particularly useful concentrations of the primersset forth in Table 1 for multiplex PCR amplification using theexperimental conditions set forth in Table 3. TABLE 2 PrimerConcentrations in p53 Primer Set Values of X  2 Typical values of C_(L)450 nM Amplicon Length Primer Concs Longest 4 368 bp 450.0 nM 5 272 bp245.8 nM 8 241 bp 193.0 nM 11 231 bp 177.3 nM 10 210 bp 146.5 nM 6 204bp 138.3 nM 7 175 bp 101.8 nM 2 164 bp  89.4 nM 9 146 bp  70.8 nMShortest 3  90 bp  26.9 nM

[0028] TABLE 3 Multiplex PCR Start with 250 ng of Template DNA. PCRComponents for 100 ul PCR in 0.2 ml thin walled tubes: Stock Conc FinalConc for 1 reaction Buffer (No Mg) 10 X  1 X  10.0 ul MgCl₂ 25 mM  2.5mM  10.0 ul dATP 10 mM 200 uM  2.0 ul dCTP 10 mM 200 uM  2.0 ul dGTP 10mM 200 uM  2.0 ul dTTP 10 mM 200 uM  2.0 ul Taq GOLD  5 U/ul  10 U  2.0ul p53 Primer Set 20 X  1 X  5.0 ul Water Human genomic 250 ng DNA TotalVolume 100.0 ul Final Concentrations in Buffer (No Mg) are 10 mMTris-HCl (pH 8.3), 50 mM KCl Taq GOLD is AmpliTaq Gold ™ from PerkinElmer catalog #N808-0243 PCR Cycles: 35 Cycles: 94° C. 10 min 94° C. 30sec 60° C. 30 sec 72° C. 45 sec 72° C. 10 min To visualize amplicons bygel Analysis: Visualize PCR products on 4% NuSieve Agarose Gel NuSieve ™Agarose 3:1 is from FMC catalog #50092 Load 5 ul of PCR + loading bufferUse 50 bp Ladder (Gibco/BRL catalog #10416-014) as size marker Run gelat 125 Volts for 30 min. to 90 min. Expected PCR Products: Order in Gel:Amplicon Length Amplicon Length Exon 2 164 bp Exon 4 368 bp Exon 3  90bp Exon 5 272 bp Exon 4 368 bp Exon 8 241 bp Exon 5 272 bp Exon 11 225bp Exon 6 204 bp Exon 10 210 bp Exon 7 175 bp Exon 6 204 bp Exon 8 241bp Exon 7 175 bp Exon 9 146 bp Exon 2 164 bp Exon 10 210 bp Exon 9 146bp Exon 11 225 bp Exon 3  90 bp

[0029] Using the methods and reagents provided herein, we achievedmultiplex PCR amplification of coding regions shown of the human p53gene in approximately equimolar amounts. That desirable result wasachieved in a single-tube reaction. The achievement of such desirableresults with the remarkable convenience of a single tube reactionfurther illustrates the contribution to the art made by the presentinvention.

[0030] The methods and compositions of the present invention are usefulin virtually any context in which equimolar yields of various PCRproducts are desired. Such contexts include without limitation paternitytesting, forensic analysis, genetic screening, polymorphism detection,and mutation analyses. The present invention can be used to amplifynucleic acids for all forms of sequence analysis known to those skilledin the art. Sequence analysis techniques includes, for example,dideoxy-sequencing and sequence analysis using high-density nucleic acidarrays: the GeneChip® probe arrays or VLSIPS™ technology of Affymetrix,Inc. High density nucleic acid arrays are discussed for example in Chee,M., Yang, R., Hubbell, E., Berno, A., Huang, X. C., Stern, D., Winkler,J., Lockhart, D. J., Morris, M. S.,, & Fodor, S. P., Science 5287,610-614 (1996), U.S. Pat. No. 5,445,934, and International PublicationNo. WO 95/11995 corresponding to PCT Application No. PCT/US94/12305.

[0031] The p53 gene and its protein product are discussed in MolecularBiology of the Cell, 3rd Edition, Alberts, B., Bray, D., Lewis, J.,Raff, M., Roberts, K., and Watson, J. D., Garland Publishing (1994) atpages 889 and 1284-1289.

[0032] It is understood that the examples and embodiments describedherein are for illustrative purposes only and that various modificationsor changes in light thereof will be suggested to persons skilled in theart and are to be included within the spirit and purview of thisapplication and scope of any appended claims. All publications, patents,and patent applications cited herein are hereby incorporated byreference in their entirety for all purposes.

1 20 1 28 DNA homo sapiens 1 tcatgctgga tccccacttt tcctcttg 28 2 28 DNAhomo sapiens 2 tggcctgccc ttccaatgga tccactca 28 3 30 DNA homo sapiens 3aattcatggg actgactttc tgctcttgtc 30 4 26 DNA homo sapiens 4 tccaggtcccagcccaaccc ttgtcc 26 5 30 DNA homo sapiens 5 gtcctctgac tgctcttttcacccatctac 30 6 26 DNA homo sapiens 6 gggatacggc caggcattga agtctc 26 728 DNA homo sapiens 7 cttgtgccct gactttcaac tctgtctc 28 8 27 DNA homosapiens 8 tgggcaacca gccctgtcgt ctctcca 27 9 29 DNA homo sapiens 9ccaggcctct gattcctcac tgattgctc 29 10 27 DNA homo sapiens 10 gccactgacaaccaccctta acccctc 27 11 27 DNA homo sapiens 11 gcctcatctt gggcctgtgttatctcc 27 12 28 DNA homo sapiens 12 ggccagtgtg cagggtggca agtggctc 2813 30 DNA homo sapiens 13 gtaggacctg atttccttac tgcctcttgc 30 14 29 DNAhomo sapiens 14 ataactgcac ccttggtctc ctccaccgc 29 15 30 DNA homosapiens 15 cacttttatc acctttcctt gcctctttcc 30 16 30 DNA homo sapiens 16aactttccac ttgataagag gtcccaagac 30 17 29 DNA homo sapiens 17 acttacttctccccctcctc tgttgctgc 29 18 31 DNA homo sapiens 18 atggaatcct atggctttccaacctaggaa g 31 19 29 DNA homo sapiens 19 catctctcct ccctgcttctgtctcctac 29 20 30 DNA homo sapiens 20 ctgacgcaca cctattgcaa gcaagggttc30

1. A method of performing multiple polymerase chain reactions in asingle vessel, comprising: priming DNA synthesis on a template in avessel with at least two sets of primers, wherein the primers arepresent in the vessel at a predetermined ratio, wherein the ratio isdescribed by: C _(A) =C _(L)(L _(A) ÷L _(L))² wherein C_(A) is theconcentration of primers for an amplicon A; wherein C_(L) is theconcentration of primer for the longest amplicon; wherein L_(A) is thelength of the amplicon A; and wherein L_(L) is the length of the longestamplicon:
 2. The method of claim 1 wherein the template is genomic DNAencoding p53.
 3. The method of claim 1 wherein the template is a cDNAencoding p53.
 4. The method of claim 1 wherein the primers amplify atleast 2 exons of p53 selected from the group consisting of exons 2-11.5. The method of claim 1 wherein the primers amplify at least 4 exons ofp53 selected from the group consisting of exons 2-11.
 6. The method ofclaim 1 wherein the primers amplify exons 2-11 of p53.
 7. The method ofclaim 4 wherein the primers are selected from those shown in SEQ ID NO:ID NOS: 1-20.
 8. The method of claim 5 wherein the primers are selectedfrom those shown in SEQ ID NO: ID NOS: 1-20.
 9. The method of claim 6wherein the primers are shown in SEQ ID NO: ID NOS: 1-20.
 10. The methodof claim 9 wherein the primers are present in the following ratios: exon2 (89.4): exon 3 (26.9): exon 4 (450): exon 5 (245.8): exon 6 (138.3):exon 7 (101.8): exon 8 (193.0): exon 9 (70.8): exon 10 (146.5): exon 11(177.3).
 11. A method of performing multiple polymerase chain reactionsin a single vessel, comprising: priming DNA synthesis on a genomic p53template in a vessel with ten sets of primers which amplify exons 2-11of p53, wherein the primers are shown in SEQ ID NOS: 1-20, wherein theprimers are present in the vessel at the following ratios: exon 2(89.4), exon 3 (26.9), exon 4 (450), exon 5 (245.8), exon 6 (138.3),exon 7 (101.8), exon 8 (193.0), exon 9 (70.8), exon 10 (146.5), exon 11(177.3).
 12. A kit comprising a set of primers for performing multiplepolymerase chain reactions in a single vessel, comprising: twentyprimers having sequences as shown in SEQ ID NO: ID NOS: 1-20.
 13. Thekit of claim 12 wherein the ratio of the concentrations of the primersis described by: C _(A) =C _(L)(L _(A) ÷L _(L))² wherein C_(A) is theconcentration of primers for an amplicon A; wherein C_(L) is theconcentration of primer for the longest amplicon; wherein L_(A) is thelength of the amplicon A; and wherein L_(L) is the length of the longestamplicon.
 14. The kit of claim 12 wherein the ratio of the primers is:exon 2 (89.4): exon 3 (26.9): exon 4 (450): exon 5 (245.8): exon 6(138.3): exon 7 (101.8): exon 8 (193.0): exon 9 (70.8): exon 10 (146.5):exon 11 (177.3).
 15. A mixture of primers for performing multiplexpolymerase chain reaction, wherein the primers are present in themixture at a predetermined ratio to each other, wherein the ratio of theconcentrations of the primers is described by: C _(A) =C _(L)(L _(A) ÷L_(L))² wherein C_(A) is the concentration of primers for an amplicon A;wherein C_(L) is the concentration of primer for the longest amplicon;wherein L_(A) is the length of the amplicon A; and wherein L_(L) is thelength of the longest amplicon.
 16. The mixture of claim 15 whichcomprises at least 4 primers.
 17. The mixture of claim 15 whichcomprises at least 6 primers.
 18. The mixture of claim 15 whichcomprises at least 8 primers.
 19. The mixture of claim 15 whichcomprises at least 10 primers.
 20. The mixture of claim 15 whichcomprises at least 12 primers.
 21. The mixture of claim 15 whichcomprises at least 14 primers.
 22. The mixture of claim 15 whichcomprises at least 16 primers.
 23. The mixture of claim 15 whichcomprises at least 18 primers.
 24. The mixture of claim 15 whichcomprises at least 20 primers.